Cytotoxic T-Lymphocyte Associated Antigen-4 (+49A/G) Gene Polymorphism as a Protective Factor against Toxoplasmosis

Risala Hussain Allami
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Keywords : Toxoplasmosis, CTLA-4+49A/G, single nucleotide polymorphism
Medical Journal of Babylon  14:2 , 2017 doi:1812-156X-14-2
Published :15 October 2017


Cytotoxic T-Lymphocyte Associated Antigen-4 (CTLA-4) is a key factor in immune regulation. Polymorphisms in CTLA-4 gene may influence the status of this factor and eventually the immune response of host against the infectious agents. This response becomes of particular importance in cases when the pathogen is an opportunistic such as Toxoplasma gondii. This study aimed to explore the effect of CTLA-4+49A/G single nucleotide polymorphism (SNP) on the susceptibility to toxoplasmosis in women. Genomic DNA was isolated from 59 women with toxoplasmosis and aged matched 60 Toxoplasma-free women as controls. Tetra-Primer Amplification Refractory System-Polymerase Chain Reaction (ARMS-PCR) was used for amplification and genotyping of CTLA-4 gene using specific primers. The heterozygous genotype (AG) and G allele of the polymorphism CTLA-4+49A/G were less frequent among cases (17.12% and 16.95% respectively) than controls (40% and 35% respectively) with significant differences (OR=0.379, 95%CI=0.206-0.697, P=0.002). These data strongly suggested the protective role of CTLA-4+49G against toxoplasmosis among Iraqi women.


Despite the worldwide prevalence of infection with Toxoplasma gondii, the disease (toxoplasmosis) rarely becomes symptomatic unless the host suffers from immunodeficiency [1]. This fact reflects the crucial importance of immune system against toxoplasmosis. Immune system does has a significant role against all infectious agents, but some of these, which are called opportunistic microorganisms such as T. gondii, usually exhibit a clinical significant only in immune-compromised hosts [2]. In many cases, immune deficiency statuses are easily recognized such as agamma-globulinemia and acquired immune-deficiency syndrome (AIDS). In other cases, there is no clear distinction, and even the term “deficiency” cannot be applied. Cases such as those associated with single nucleotide polymorphisms (SNPs) affecting the immune response could be placed within this category. For instance, SNPs in vitamin D receptor gene was reported to be significantly associated with toxoplasmosis among Iraqi women [3]. Cytotoxic-T lymphocyte-associated antigen 4 (CTLA-4) is an inhibitory receptors expressed transiently especially on CD4+ and CD8+ T cell and constitutively on CD4+CD25+ T-regulator (T-reg) cells [4]. This receptor has a decisive function in fine regulation of T-cell response by exerting a negative signals for T-cell activation by interacting with its ligands, B7.1 and B7.2 on antigen presenting cells (APCs) [5]. The CTLA-4 gene is located on chromosome 2q33 and it consists of four exons and three introns. This gene encodes for two different protein forms: soluble CTLA-4 (sCTLA-4) and full length CTLA-4 (flCTLA-4) with the former is lacking the exon 3 [6]. Approximately 100 SNPs have been reported in this gene; however only two as well as one microsatellite of which are found to be significantly associated with certain diseases. These are CTLA-4+49A/G (rs231775), CTLA-4?318C/T (rs5742909) and microsatellite (AT)n repeat in the 3’-untranslated region (3’UTR) [7]. Previous reports have elucidated the effect of CTLA-4+49A/G with three kinds of diseases: autoimmune diseases such as graves’ disease [8], Hashimoto thyroiditis [9], autoimmune hypothyroidism [10], systemic lupus erythematosus, celiac disease, Henoch-Schnlein purpora and type 1 diabetes mellitus [9,11,12]; cancers like breast, lung, esophageal, gastric, colorectal, oral, cervical, and renal cell carcinoma [13,14,15,16]; and finally with few infectious diseases particularly tuberculosis, visceral leishmaniasis and hepatitis B infection [17,18,19]. This study aimed to assess the effect of this SNP on Iraqi women susceptibility to develop active toxoplasmosis. To the best of our knowledge, there is no previous published work addressed such exploration.

Materials and methods

Women attending the Department of Obstetrics and Gynecology at Al-Imamain Al-Kadhimain Medical City/ Baghdad from January, 2015 to October, 2016 were eligible for this study. The age range was 22-49 years, mean= 32.19 years. Those women were suffering from different gynecological diseases. From each woman, 5 mL of venous blood were obtained and divided into two parts. The first part (3ml) was in aplain tube from which serum was obtained. The second part (2ml) was placed in EDTA tube for DNA isolation.
For detection of anti-toxplasma antibodies, two laboratory method were used.. Those were rapid test cassette (CTK Biotech Inc., USA), and E for LISA IgG and IgM antibodies (Cusabio/China). According to these methods, 59 women out of 342 tested gave positive results for toxoplasmosis. Sixty women from those gave negative result were randomly chosen to be control.

DNA Isolation and Polymerase Chain Reaction
DNA was isolated from whole blood using a ready kit (ZymoBead™ Genomic DNA Kit, USA) according to manufacturer’s protocol. Tetra-Primer Amplification Refractory Mutation System (ARMS-PCR) method was used for CTLA-4 gene amplification with four primers (Table 1)].


Characteristics of the Study Population Table 2 shows the demographic characteristics of the study population. According to results of this table, there are no significant differences in term of age, parities, body mass index (BMI) or dwelling between toxoplasma-infected women and toxoplasma-free women.


So far, this is the first published study exploring the effect of CTLA-4+49A/G polymorphism on the susceptibility to toxoplasmosis in Iraq. The study revealed significant protective role of AG genotype (OR=0.146, 95% CI=0.029-0.730, P= 0.019) and G allele (OR= 0.379, 95% CI=0.206-0.697P=0.002) against the disease. These results are in line with that obtained by Hajilooi et al. [18] in term of leishmaniasis, Paad et al. [17] regarding tuberculosis, and Xu et al. [19] in hepatitis B infection. All these microorganisms share Toxoplasma with their intracellular residency. However, Lazano et al. [21]did not find any significant effect of this polymorphism on the incidence of the fungal disease, paracoccidioidomycosis, among Brazilian population. These disparities may be due to the apparent heterogeneity between different populations, and to the influence of environmental factors affecting different diseases. The CTLA-4+49A/G polymorphism involves the substitution at the site 49 in CTLA-4 gene of adenine with guanine. Accordingly, the codon 17 (ACC) which encodes threonine is substituted by GCC which encodes alanine. The CTLA-4 receptor achieves essential regulatory function during immune response by controlling the overall strength of T-cell activation [22]. In fact, two mechanisms have been postulated for this regulatory effect. The first one is interacting of CTLA-4 with its ligands B7.1 and B7.2 depriving the homologue receptor CD28 from their ligands, while the second mechanism is the inhibition of T-cell activation through signal transduction pathway which down-regulates the T-cell receptor dependent signaling [23]. Substitution of threonine by alanine results in many phenotypic changes affecting one or both of these two mechanisms. It was postulated that alanine-containing CTLA-4 protein suffers from an altered spatial configuration which causes a fault in handling of this protein in the endoplasmic reticulum with less efficient N-glyco-sylation [24]. This glycosylation is very important in the dimerization and then the triggering of inhibitory function of CTLA-4 [25]. Furthermore, some evidence suggested a significant decrease in mRNA for CTLA-4 protein associated with GG genotype in patients with autoimmune diseases [26]. Sun et al. [27] suggested that G allele do not only cause down-regulation in the production of CTLA-4 but also results in a protein with lower affinity to B7. Regardless of the mechanism by which G allele-bearing CTLA-4 affecting the immune response, there is almost a general agreement that this variant has less ability to control the activation of T-cells compared to A allele-bearing CTLA-4. But this is a double-edge sword. From one edge, the +49G variant carriers have the merit of robust immune response with long lasting T-cell activation, and this hypothetically protects them from different infectious agents and may be some malignancies. However, from the other edge this variant predispose its carriers to wide range of autoimmune diseases such as Graves’ disease and type 1 diabetes mellitus. In conclusion, G allele of the SNP CTLA-4+49A/G appears to have a protective role against toxoplasmosis in Iraqi women. Further studies with a larger sample and different ethnic population are required for more solid conclusion.


1- Robert-Gangneux F, Dardé ML. Epidemio-logy of and diagnostic strategies for toxoplasmosis. Clin. Microbiol. Rev., 2012, 25:264–96.
2- Zhou P, Chen Z, Li H, Zheng H, He S, Lin R, et al. Toxoplasma gondii infection in humans in China. Parasites Vectors, 2011,4:165.
3- Al-Mayah QS. The Impact of Vitamin D Receptor Gene Polymorphisms and Haplotypes on the susceptibility to Toxoplasmosis. Dyiala J Med 2017; 2(1):1-8.
4- Balbi G, Ferrera F, Rizzi M, Piccioli P, Morabito A, Cardamone L,et al. Association of -318 C/T and +49 A/G cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphisms with a clinical subset of Italian patients with systemic sclerosis. Clin Exp Immunol 2007;149:40-47.
5- Chen L. and Flies DB. Molecular mechanisms of T cell co-stimulation and co-inhibition. Nat Rev Immunol 2013, 13(4): 227-242.
6- Oaks MK and Hallett KM. Cutting edge: a soluble form of CTLA-4 in patients with autoimmune thyroid disease. J Immunol 2000; 164(10):5015-5018.
7- Kouki T, Gardine CA, Yanagawa T. and Degroot LJ. Relation of three polymorphisms of the CTLA-4 gene in patients with Graves disease. J Endocrinol Invest 2002; 25(3):208–813.
8- Du F, Ma X. and Wang C. Association of CTLA4 gene polymorphisms with garves’ ophthalmopathy: a meta-analysis. Int J Genomics 2014;2017:ID 537969.
9- Vaidya B. and Pearce S. The emerging role of the CTLA-4 gene in autoimmune endocrinopathies. Eur J Endocrinol 2004; 150(5):619-626.
10- Patel H, Mansuri MS, Singh M, Begum R, Shastri M. and Misra A. Association of Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) and Thyroglobulin (TG) Genetic Variants with Autoimmune Hypo-thyroidism. PLoS One2016; 11(3): e0149441.
11- Hunt KA, McGovern DPB, Kumar PJ, Ghosh S, Travis SP, Walters JR. et al. A common CTLA4 haplotype associated with coeliac disease. Europ J Human Genetics 2005; 13(4)440–444.
12- Wang JJ, Shi YP, Yue H, Chun W. and Zou LP. CTLA-4 exon 1 +49A/G polymorphism is associated with renal involvement in pediatric Henoch-Sch?nlein purpura. Pediatric Nephrol2012;27(11): 2059–2064.
13- Bharti V, Mohanti BK and Das SN. Functional genetic variants of CTLA-4 and risk of tobacco-related oral carcinoma in high-risk North Indian population. Hum Immunol 2013,74:348–352
14- Xiaolei L, Baohong Y, Haipeng R, Shuzhen L, Jianfeng G, Xiangpo P. et al. Current evidence on the cytotoxic T-lymphocyte antigen 4 + 49G > A polymorphism and digestive system cancer risks: a meta-analysis involving 11,923 subjects. Meta Gene 2015,6:105-108.
15- Hu L, Liu J, Chen X, Zhang Y, Liu L, Zhu J. et al. CTLA-4 gene polymorphism +49 A/G contributes to genetic susceptibility to two infection-related cancers-hepato-cellular carcinoma and cervical cancer. Hum. Immunol 2010,71:888–891.
16- Dai Z, Tian T, Wang M, Liu X, Lin S, Yang P et al. CTLA-4 polymorphisms associate with breast cancer susceptibility in Asians: a meta-analysis. Peer J 2017;5: e2815.
17-Paad E, Tamendani MK, Sangtarash MH and Dehvari M. Analysis of CTLA-4 (+49A/G) gene polymorphism and the risk of tuberculosis in Southeast of Iran. Gene Cell Tissue2014;1(13):e23996.
18- Hajilooi M, Lotfi P, Seif F, Bazmani A, Momeni M, Ravary Aet al. The cytotoxic T lymphocyte antigen-4 +49A/G single nucleotide polymorphism association with visceral leishmaniasis. Jundishapur J Microbiol 2013;7(10): e12143.
19- Xu H, Zhao M, He J and Chen Z. Association between cytotoxic T-lymphocyte associated protein 4 gene +49 A/G polymorphism and chronic infection with hepatitis B virus: a meta-analysis. J Int Med Res 2013;41(3):559-567.
20- Noorie-Nejad M, Taji O, Tamandani DM and Kaykhaei MA. Association of CTLA-4 gene polymorphisms?318C/T and +49A/G and Hashimoto s thyroidits in Zahedan, Iran. Biomed Rep 2017; 6(1): 108-112.
21- Lazano VF, Lins TC, Teixeira MM, Vieira RG, Blotta MH. Goes AM et al. Polymorphism analysis of CTLA-4 gene in paracoccidioidomycosis patients. MemInst Oswaldo Cruz 2011; 106(2):220-226.
22- Bour-Jordan H, Grogan JL, Tang Q, Auger JA, Locksley RM and Bluestone JA. CTLA-4 regulates the requirement for cytokine induced signals in Th2 lineage commitment. NatImmunol 2003; 4(2): 182– 188.
23-Baroja ML, Darlington PJ, Carreno BM and Madrenas J. Inhibition of T cell activation by CTLA-4: truths and red herrings. Mod Asp Immunobiol 2000;1: 169-173.
24- Pavkovic M, Georgievski B, Cevreska L, Spiroski M andEfremov DG. CTLA-4 exon 1 polymorphism in patients with autoimmune blood disorders. Am J Hematol 2003;72: 147-149.
25-Darlington PJ, Kirchhof MG, Criado G, Sondhi J and Madrenas J. Hierarchical regulation of CTLA-4 dimer-based lattice formation and its biological relevance for T cell inactivation. JImmunol 2005, 175(2): 996-1004.
26- Daroszewski J, Pawlak E, Karabon L, Frydecka I, Jonkisz A, Slowik M et al. Soluble CTLA-4 receptor an immune-logical marker of Graves disease and severity of ophthalmopathy is associated with CTLA4 Jo31 and CT60 gene polymorphisms. Eur J Endocrinol 2009; 161(5):787–793.
27- Sun T, Zhou YF, Yang M, Hu Z, Tan W, Han X et al. Functional genetic variations in cytotoxic T-lymphocyte antigen 4 and susceptibility to multiple types of cancer. Cancer Res 2008; 68(17): 7025-7034.

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